Protein interactions facilitate most biological processes including signal transduction and homeostasis. The elucidation of particular interacting protein partners facilitating these biological processes has been advanced by the development of in vivo “two-hybrid” or “interaction trap” methods for detecting and selecting interacting protein partners (see Fields & Song (1989) Nature 340: 245–6; Gyuris et al. (1993) Cell 75: 791–803; U.S. Pat. No. 5,468,614; and Yang et al. (1995) Nucleic Acid Research 23, 1152–1156). These methods rely upon the reconstitution of a nuclear transcriptional activator via the interaction of two binding partner polypeptides—i.e. a first polypeptide fused to a DNA binding domain (BD) and a second polypeptide fused to a transcriptional activation domain (AD). When the first and the second polypeptides interact, the interaction can be detected by the activation of a reporter gene containing binding sites for the DNA binding domain. For this method to work, both proteins need to be soluble and must be able to localized to the nucleus. Accordingly, the interaction of polypeptides which are normally localized to other compartments may not be detected because of the absence of other non-nuclear polypeptide components which facilitate the interaction or particular non-nuclear post-translational modifications which fail to occur in the nucleus or because the interacting proteins fail to fold properly when localized to the nuclear compartment. In particular, the nuclear two-hybrid assay is ill-suited to the detection of protein interactions occurring within or at the surface of cellular membranes. In addition, this assay is unsuited for screening small molecule-protein interactions because it relies solely on genetically encoded fusion proteins.
A fundamental area of inquiry in pharmacology and medicine is the determination of ligand-receptor interactions. The pharmacological basis of drug action, at the cellular level, is quite often the consequence of non-covalent interactions between therapeutically relevant small organic molecules and high affinity binding proteins within a specific cell type. These small organic ligands may function as agonists or antagonists of key regulatory events which orchestrate both normal and abnormal cellular functions. For years the pharmaceutical industry's approach to discovering such ligands has been one of the random screening of thousands of small molecules in specific in vitro and in vivo assays to determine a potent lead compound for their drug discovery efforts. Using these tools, a lead compound may be found to exert very well-defined effects with regard to a function in one particular cell type (e.g. inhibition of cytokine production or DNA replication in a particular cancer cell line). However, such results may give little indication as to the mechanism of action at the molecular (ligand-protein interaction) level. Furthermore, the screening for potent action on one cellular function may miss out on cross-reactivities of a lead compound giving rise to undesired side-effects. Such side-effects often are the consequence of proteins with closely similar structures having different functions, or of a protein fulfilling different functions when expressed in different cell types, or even when localized to different sub-cellular compartments. Therefore, the identification of the possibly various protein targets for a pharmacological agent displaying a given activity is challenging but highly desirable. There is an unmet need for a general and efficient method to identify the cellular targets for these pharmacological agents so as to accelerate the search for novel drugs both at the basic and applied levels of research.
Similarly, there is a need for a general approach to identify a small molecule capable of binding any selected cellular target regardless of its biological function. Fowlkes et al. (WO 94/23025) and Broach et al. (WO 95/30012) described a screening assay for identifying molecules capable of binding cell surface receptors so as to activate a selected signal transduction pathway. These references describe the modification of selected yeast signaling pathways so as to mimic steps in the mammalian signaling pathway. This latter approach is specific for certain signaling pathways and has limited utility for broadly discovering small molecules that interact with any cellular target. Thus, there is also an unmet need for a general screening method to determine the interaction between small molecules and target proteins so as to identify new drugs that are capable of specific therapeutic effects in a variety of disease states as well as to identify agonists and antagonists that may interfere or compete with the binding of the small molecules for these targets.
At this time, few (if any) efficient methodologies exist for rapidly identifying a biological target such as a protein for a particular small molecule ligand. Existing approaches include the use of affinity chromatography, radio-labeled ligand binding and photoaffinity labeling in combination with protein purification methods to detect and isolate putative target proteins. This is followed by cloning of the gene encoding the target protein based on the peptide sequence of the isolated target. These approaches require substantial re-development of matrices and the conditions of their use for each ligand under investigation, and are therefore laborious and painstaking.
Crabtree et al. (WO 94/18317) described a method to activate a target gene in cells comprising (a) the provision of cells containing and capable of expressing (i) at least one DNA construct comprising at least one receptor domain, capable of binding to a selected ligand, fused to a heterologous additional protein capable of initiating a biological process upon exposure of the fusion construct to the ligand, wherein the biological process comprises the expression of the target gene, wherein the ligand is capable of binding to two or more fusion proteins, and wherein the biological process is only initiated upon binding of the ligand to two or more fusion proteins, the two fusion proteins being the same or different, and (ii) the target gene under the expression control of a control element which is transcriptionally responsive to the initiation of said biological process; and (b) exposing said cells to said ligand in an amount effective to result in expression of the reporter gene. Further described are DNA constructs, ligands and kits useful for performing such method. Related documents U.S. Pat. No. 5,830,462, U.S. Pat. No. 5,869,337 U.S. Pat. No. 6,165,787 show these and other embodiments; specifically, Holt et al. (WO 96/06097) describes the synthesis of hybrid ligands for use with the subject methods. The purpose envisaged for these methods and compositions is restricted to the investigation of cellular processes, the regulation of the synthesis of proteins of therapeutic or agricultural importance and the regulation of cellular processes in gene therapy. Nothing therein suggests the use of these methods and compositions to study the interaction of proteins with small molecules, particularly in its application to pharmaceutical research and drug development.
Licitra and Liu (WO 97/41255) described a “three hybrid screen assay” in which the basic yeast two-hybrid assay system is implemented. The significant difference is: instead of depending on the interaction between a so-called “bait” and a so-called “prey” protein, the transcription of the reporter gene is conditioned on the proximity of the two proteins, each of which can bind specifically to one of the two moieties of a small hybrid ligand. The small hybrid ligand constitute the “third” component of the hybrid assay system. In that system, one known moiety of the hybrid ligand will bind to the “bait” protein, while the interaction between the other moiety and the “prey” protein can be exploited to screen for either a protein that can bind a known moiety, or a small moiety (pharmaceutical compound or drug) that can bind a known protein target.
However, the three hybrid system of Liu suffers from several limitations: 1) the use of a transcriptional activation reporter assay is ill-suited for non-nuclear proteins, for example, membrane-bound proteins and cytosolic proteins; 2) the hybrid ligand must be localized to the nucleus, and remains stable; and, 3) the interaction between the “bait” protein and its binding moiety on the hybrid ligand must have high affinity, preferably at the nanomolar level. For example, FK506-FKBP interaction was used which provides micromolar affinity. Higher affinity bewteen bait protein and its binding partner is desired for improving system performance.
Lin et al. (J. Am. Chem. Soc. 2000, 122:4247–8) improved upon the existing three hybrid system by replacing the FK506-FKBP pair with a hybrid ligand consisting of dihydrofolate-reductase (DHFR) linked to methotrexate (Mtx) (DHFR-Mtx), which provides picomolar affinity, thereby significantly improving system performance.
U.S. Pat No. 5,585,245 and U.S. Pat. No. 5,503,977 describe the “split ubiquitin” methods, which can detect protein-protein interactions by use of a ubiquitin specific protease to cleave a reporter polypeptide from a fusion protein. Two fusion proteins are constructed, one consisting of the N-terminal half of ubiquitin and a prey protein (Nub-prey or prey-Nub), and the other consisting of the C-terminal half of ubiquitin, a bait protein and the reporter (bait-Cub-reporter). Association of prey and bait reconstitutes a ubiquitin structure recognized by the ubiquitin specific protease, whereby the reporter is cleaved from the fusion protein. The cleavage of the reporter from the fusion protein can be detected by several techniques, e.g. cleavage or destabilizing the reporter or allow for its translocation.
A further working principle used in several assay systems developed to investigate protein-protein interactions is the reconstitution of an enzymatic activity from the induced spatial proximity of enzymatic fragments mediated by the interaction of two peptides fused to these fragments. Such an assay is termed an enzyme complementation assay. U.S. Pat. No. 6,270,964, WO 98/44350 and Wehrman et al., Proc. Natl. Acad. Sci. U.S.A. (2002), 99:3469–3474, show exemplary methods employing this principle.
WO 93/08278, WO 98/37186, WO 01/14539 and WO 02/22826 describe yet another biological system for the investigation of protein-protein interactions. Therein, genetic information encoding the peptides or proteins to be tested for interactions is cloned into a vector comprising genetic information encoding a nucleic acid binding protein as well as the nucleic acid sequence said nucleic acid binding protein binds to, such that the peptides or proteins are expressed as in-frame fusions with the nucleic acid binding domain. When cells are induced to express the fusion peptides/proteins, they will associate with the vector that encodes them. After isolation of these complexes from the cells and testing for interaction, the nucleic acid encoding interacting peptides/proteins is easily retrieved. WO 98/37186, WO 01/14539 and WO 02/22826 particularly describe systems wherein the nucleic acid binding protein forms a covalent bond with its recognition motif.
So called “pull-down” techniques are still frequently used in the investigation of protein-protein interactions. As opposed to the methods described above, these methods are carried out in vitro rather than in vivo. In essence, these methods rely on immobilizing the molecular species for which a binding or interaction partner is sought on a surface, and subsequently passing a solution containing potential binding partners/interactors over this surface. A binding/interaction partner will be retained on the solid support, while other constituents of the solution will be washed away. In a second step, the binding/interaction partner is isolated for further analysis, for example by passing a solution containing an excess of a substance known to competitively displace binding/interaction partners from the molecular species under investigation. Alternatively, the bond between the molecular species under investigation and the matrix may be severed and the complex isolated from the solid support for analysis. An example of the use of such a technique to identify intracellular targets of purvalanol B, an inhibitor of CDKs, is shown in Knockaert et al. (2000), Chem. Bio. 7:411–422.